Patent Application
20150259724
This invention relates generally to detecting biomarkers and, more particularly, to an apparatus and method for detecting and/or characterizing proteolytic enzymes or other biomarkers for a wide variety of applications, such as common diseases, infectious diseases, cancer, food safety, biodefense, etc.
A biomarker, or biological marker, generally refers to a measurable indicator of some biological state or condition. Certain biochemical components, such as enzymes, proteins, nucleic acids, lipids, etc., can be used as biomarkers for the presence of the biological entity or condition (e.g., virus, bacteria, damaged cell, etc.) that created the biomarkers. The detection of the biomarker component can be used to detect the presence of the producing entity. As an example, a proteolytic enzyme, also referred to as a protease, peptidase, or proteinase, is an enzyme that performs proteolysis, or protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein. The presence of particular proteolytic enzymes can be indicators of particular conditions. As a specific example, the HIV-1 protease is a retroviral aspartyl protease, and has been recognized as an essential element in maturation of the infectious virus. Accordingly, HIV-1 protease has been an important target for drug therapy.
At present, the majority of the developed methods for HIV detection are based on the detection of the presence of antibodies that the patient's body makes against HIV, direct molecular recognition of HIV and its components such as specific nucleic acid sequences or antigens, or measurement of the activity of HIV-1 protease, many of which are often laborious and time-consuming, and/or require the use of labels or sophisticated instruments.
There is a continuing need for improved detection of common and/or infectious diseases or conditions.
A general object of the invention is to provide an apparatus and method for detecting biomarkers, such as proteins, enzymes or nucleic acids (RNA or DNA), for a wide variety of applications, such as common diseases, infectious diseases, cancer, food safety, pharmaceutical screening, environmental protection, biodefense, etc.
The general object of the invention can be attained, at least in part, through a method for sensing or characterizing biomarkers, such as proteins, enzymes or nucleic acids. The method includes providing two fluid compartments separated by an electrically resistant membrane bilayer including a nanopore, introducing a sample, such as containing a substrate or nucleic acid probe, to a first fluid compartment, and applying an electric field across the membrane. A current modulation signature of the sample is monitored across the membrane, from which a presence of a biomarker in the sample can be determined as a function of the sample current modulation signature. If the biomarker is present, the target substrate or nucleic acid probe within a first of the fluid compartments is modified, such as by enzyme cleavage or hybridization, resulting in a change in the ionic current passing through the nanopore. The presence of the biomarker can be determined by comparing the sample current modulation signature to a known substrate current modulation signature for the non-cleaved polypeptide substrate and/or sample.
The invention further includes a system for sensing or characterizing a proteolytic enzyme. The system comprises a nanopore sensor to determine a current modulation of a sample including a polypeptide substrate, and a predetermined substrate current modulation signature for comparison to a current signature from the nanopore sensor. The nanopore sensor includes two fluid compartments, with a membrane separating the first fluid compartment and the second fluid compartment. A nanopore through the membrane fluidically connects the first compartment and the second compartment, and a power supply in electrical contact with the membrane provides an electric potential difference between the fluid compartments. A detector is used to detect an electrical current through the nanopore as the polypeptide substrate, or components thereof, transits the nanopore under an applied electric potential difference between the fluid compartments.
The present invention includes a rapid, label-free method for the sensitive and accurate measurement of protease activity by real-time monitoring of the ionic current modulations arising from the substrate peptide-protease interactions in a nanopore. This method is rapid and sensitive: picomolar concentrations of protease can be accurately detected in approximately 10 minutes. Further, the protease assay does not require the use of expensive equipment. The substrate-based nanopore sensing approach is useful for nanopore sensors of various proteolytic enzymes, such as, without limitation, HIV protease, HCV protease, metalloprotease, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), serine protease (e.g., trypsin), cysteine protease, threonine protease, aspartic protease, etc.
Embodiments of this invention provide a rapid, sensitive, and label-free nanopore sensing apparatus and method for the detection of various proteases. In embodiments of this invention, a protease target compound is utilized as the sensing element, while an unmodified peptide is used as the substrate. Under a fixed potential bias applied across the nanopore membrane, the activity of the protease can be detected and quantified by monitoring the change in the ionic current passing through the nanopore, which is due to the cleavage of the peptide by the protease. The method is sensitive (can detect protease at pico- to nanomolar levels) and selective (other proteases will not interfere with the detection).
The nanopore sensor of this invention can be incorporated in the development of cost-effective stochastic sensors for proteases of medical and biological importance. Such sensors not only have potential clinical values for diagnosis or prognosis of infectious diseases, cancer, and cardiovascular diseases, but also could be utilized for drug discovery and development.
Other objects and advantages will be apparent to those skilled in the art from the following detailed description taken in conjunction with the appended claims and drawings.
The present invention provides a real-time nanopore sensing method and system for the sensitive detection or characterization of a biomarker. The invention can be used for detection of various biomarkers, such as proteins, enzymes, nucleic acids (e.g., RNA or DNA), lipids, etc., but will be generally discussed below referring to proteolytic enzymes.
Nanopore detection is achieved by monitoring ionic current modulations produced by the passage of target analytes through one or more nanopores. Under conditions of constant electrolyte pH, temperature, and applied potential, the extent of current blockage (amplitude) is related to the size/diameter of the analyte molecule, while the event residence time depends on the length of the analyte and the strength of the interaction between the analyte and the nanopore. Since analytes pass through a nanopore at one molecule at a time, the event frequency can be correlated with the concentration of the analyte, and simultaneous quantification of multiple components in a solution mixture can be readily accomplished using a single nanopore as long as the nanopore itself can provide enough resolution.
The invention provides methods and systems for detecting protease activity. In addition to monitoring peptide-protease interaction, the invention can be used to obtain quantitative chemical kinetics information on the enzymatic process. The invention can also be used for recognition of peptides, including differences of one or more amino acids. In general, with an increase in the length of the peptide, both the amplitude and residence time of the events increase. With an appropriately engineered αHL nanopore, the invention can discriminate between peptides having the same length and composition but possessing different sequences. The findings suggest that nanopore sensing technology has the potential to be utilized for protein sequencing. Another advantage of the nanopore sensing of this invention is that multiple components in a solution mixture can be quantitated simultaneously using a single nanopore because analytes interact with the nanopore at one molecule at a time. Short peptide fragments, obtained from protease cleavage of a longer substrate peptide, can thus be identified and even quantified by the nanopore sensor without the need for separation and purification.
A power supply 40 is connected in electrical contact with the sensor 30 and the membrane 36 to provide an electric potential difference between the first fluid compartment 32 and the second fluid compartment 34. The power supply 40 can connect to the sensor by electrodes 42, such as Ag/AgCl electrodes, each in electrical combination with one of the first fluid compartment 32 and the second fluid compartment 34. As shown in
A detector 50 is used to detect an electrical current through the nanopore 38 as the polypeptide substrate, or components thereof, transits the nanopore 38 under an applied electric potential difference between the first and second fluid compartments 32, 34. Under a fixed potential bias applied across the nanopore membrane 36, the activity of the protease can be detected and quantified by monitoring the change in the ionic current passing through the nanopore, which is due to the cleavage of the target analyte substrate, such as a peptide, by the protease.
According to embodiments of this invention, a sample is introduced to the first fluid compartment 32. An electric field is applied across the membrane 36, such as a fixed applied potential, to drive a target analyte substrate through the nanopore 38. By the detector 50, a current modulation signature across the membrane 36 is monitored and determined, and from the current modulation signature a presence of a proteolytic enzyme in the sample can be determined. As illustrated in
In embodiments of this invention, the polypeptide substrate can include more than one cleavage site, each cleaved by one of two or more different proteolytic enzymes. The predetermined substrate current modulation signature can correspond to cleavage products of one or both of the two different proteolytic enzymes, thereby identifying one or more of the multiple enzymes. The presence of each of the multiple different proteolytic enzymes alone and/or a combination of the two different proteolytic enzymes in the sample can be determined by comparing the sample current modulation signature to the known substrate current modulation signature. For example, take a peptide substrate containing two cleavage sites, each corresponding to one of two different proteases, if only one protease is present in the sample, the substrate will be cleaved into two fragments. Therefore, the observation of two new types of current modulations indicates the presence of one protease in the sample. Furthermore, by comparing the signatures of these new events with those of the predetermined peptide fragments, the identity of the protease can be determined. If two proteases are present in the sample, three fragments will be produced from the proteolytic cleavage of the substrate.
In one embodiment of this invention, a target analyte is added with the sample to the sensor compartment. The added analyte can be a natural substrate or a modified substrate, such as a modified or engineered polypeptide including one or more additional amino acids, for a target proteolytic enzyme. The modified polypeptide substrate can provide cleavage segments of different lengths upon contact with the proteolytic enzyme. The known cleavage segments will produce predictable current modulation signatures in the sensor, thereby indicating the presence of the enzyme in the sample.
The method and system of this invention can also be used to determine a concentration of the proteolytic enzyme and/or a polypeptide substrate. In one embodiment, the concentration can be determined by a frequency of occurrence of nanopore blockage events within the sensor. By monitoring the ionic current modulations produced by the passage of the target analyte through a single nanopore at a fixed applied potential, the concentration of the analyte can be obtained by the frequency of occurrence (1/τon) of the blockage events, while its identity can be determined from the mean residence time (τoff) of the analyte coupled with the extent of current blockage (amplitude). Under experimental conditions of constant electrolyte pH, temperature, and applied potential bias, the event blockage amplitude is related to the size, structure, and/or conformation of the analyte molecule, while the event residence time depends on the length of the analyte and the strength of the interaction between the analyte and the nanopore. In addition to biosensing, nanopore analysis can be utilized for a variety of other applications, including studying covalent and non-covalent bonding interactions, investigating biomolecular conformational changes, probing enzyme kinetics, and so on.
The method and system of this invention can be used to detect various enzymes, and can be applied for any suitable detection purposes, such as, without limitation, for medical purposes, food safety purposes, and/or biodefense purposes. As one example, the method and system can be used to detect infections and/or diseases. In one embodiment of this invention, an infection is determined by identifying the presence of proteolytic enzymes in a sample that are known to be produced by a pathogen during such an infection. In a similar embodiment, a disease can be identified by identifying the presence of proteolytic enzymes in a sample that are known to be produced by diseased cells, such as cancer cells.
In one embodiment of this invention, the method and system are used to determine the presence of a retroviral infection, such as HCV or HIV, by determining the presence of a retroviral aspartyl protease in a patient sample. As one example, the activity of HIV protease is measured by real-time monitoring of the ionic current modulations arising from the substrate-protease interactions. As shown in
In another embodiment of this invention, the method and system provide a biosensor to measure proteolytic enzymes as an indicator for disease. As an example, trypsin is a serine protease which cleaves peptide bonds after arginine or lysine amino acid residues. Patients suffering from acute pancreatitis exhibit higher concentrations of trypsin than healthy people, thereby making trypsin a useful biomarker for pancreatitis. In one embodiment of this invention, a rapid and sensitive method for the detection of trypsin is provided by using a biological alpha-hemolysin nanopore. Due to a larger molecular diameter than a narrow pore constriction, trypsin cannot transport through the alpha-hemolysin channel. Hence, an indirect detection method is developed by using a lysine-containing peptide as the substrate, and analyzing the cleavage products of the substrate after trypsin digestion.
As another example, the invention includes a nanopore sensor system for detecting cancer. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) are considered biomarkers and potential therapeutic targets in human cancers. MMPs are a family of more than 20 zinc-dependent endopeptidases that share a similar structure, and are collectively capable of degrading all components of the extracellular matrix and basement membrane. They play important roles in cell biological processes and many fundamental physiological events involving tissue remodeling, such as angiogenesis, bone development, wound healing, and mammary involution. However, dysregulated activities of MMPs may lead to a number of pathological conditions such as tumor growth, invasion, and metastasis, rheumatoid arthritis, and heart failure. ADAMs are a family of more than 30 integral membrane and secreted glycoproteins that are related to snake venom metalloproteases and MMPs. Elevated activity levels of MIVIPs and/or ADAMs have been observed in almost every type of human cancer, and found to correlate with advanced tumor stage, increased invasion and metastasis, and shortened survival. MMPs and ADAMs can serve as valuable biomarkers for early diagnosis and prognosis in human cancers, and are becoming increasingly important targets for drug discovery and cancer therapy.
Embodiments of this invention provide a sensitive, selective, cost-effective, and, optionally, multiplexed sensing platform to profile the activities of MMPs/ADAMs for early cancer detection and diagnosis. The method and apparatus can operate by measuring protease activities based upon the increase in the concentration of the target degradation products of the substrate. With this strategy provided by this invention, the generated different peptide fragments allow the differentiation between the target MMP/ADAM and false positives, resulting from the common occurrence of non-target proteases and the target MMP/ADAM cleaving the substrate peptide at different positions.
In one embodiment, a panel of MMPs/ADAMs can be detected instead of a single protease as cancer biomarkers to improve cancer diagnostic accuracy. For this purpose, the invention includes multiplex nanopore sensor platforms for the concurrent measurement of the activities of multiple MMPs/ADAMs. The ability to measure the activities of multiple MMPs/ADAMs in a single assay has many advantages over singleplex systems, including reduced assay costs, improved turnaround time, reduced sample/reagent volume, high-throughput screening, and decrease in errors between inter-sampling. An added advantage of measuring protease activities by monitoring the substrate degradation products is that multiplex detection of the activities of multiple MMPs/ADAMs can be readily accomplished by using a single nanopore and a single substrate containing multiple cleavage sites for different proteases instead of the conventional sensor array format, thus further reducing the up-front costs of the protease assays (due to fabrication and use of fewer nanopores).
Measuring the activities of MMPs/ADAMs can be achieved by real-time monitoring of the ionic current modulations caused by the protease-peptide interactions in the nanopore. In the absence of proteases, the current modulations are caused only by the substrate. However, in sharp contrast, in the presence of the target protease, the substrate will be cleaved into two or more fragments (depending on the number of the substrate cleavage sites). Since the substrate breakdown products have shorter lengths than the substrate, new blockage events having smaller residence times and/or blockage amplitudes than those of the substrate may be observed. Furthermore, with a constant initial substrate concentration and a fixed amount of reaction time, the concentration and hence the event frequency of the substrate breakdown products depend on the activity of the protease. As discussed above, in the case that other proteases can also cleave the same substrate but at different positions, the substrate cleavage site allows the target MMP/ADAM to be differentiated from other proteases based on their produced quite different substrate breakdown products and their corresponding events with different signatures, thus improving the selectivity of the protease sensor.
Embodiments of this invention include label-free, real-time nanopore sensing methods and systems for the detection of nucleic acid biomarkers (e.g., DNA or RNA) using nucleic acid probes. The method is again rapid and sensitive: picomolar concentrations of the target biomarker nucleic acid could be detected rapidly, such as in approximately 1 minute. Further, the method is selective, which can differentiate the target nucleic acid DNA from other single-stranded nucleic acid molecules at the single-base resolution. This sequence-specific detection according to this invention can be particularly useful in nanopore sensors for, without limitation, pathogens.
In embodiments of this invention, the nanopore detection of pathogens or other diseases is based on the hybridization between a characteristic single-stranded gene segment of the target pathogen and an unmodified complementary single-stranded DNA (cDNA) probe. Although their diameters are typically larger than the channel constriction, short double-stranded DNA (dsDNA) molecules can be rapidly unzipped through an appropriately engineered α-hemolysin (αHL) protein nanopore. The nanopore can be engineered with functional groups, such as by introducing positively charged or aromatic groups into the nanopore, to facilitate unzipping of dsDNA.
As shown in
The invention provides or includes probes that form blunt-end ds-DNA with the target DNA, without less desirable overhangs. In one embodiment, the nucleic acid probe will form relatively long (e.g., 20 bp) full-matched blunt-ended dsDNA with the target nucleic acid biomarker. The method is thus more accurate, which is important in medical diagnosis.
The present invention is described in further detail in connection with the following examples which illustrate or simulate various aspects involved in the practice of the invention. It is to be understood that all changes that come within the spirit of the invention are desired to be protected and thus the invention is not to be construed as limited by these examples.
A nanopore sensing apparatus for the detection of HIV-1 protease was created, in which an engineered alpha-hemolysin was utilized as the sensing element, while an unmodified peptide was used as the substrate. Under a fixed potential bias applied across the nanopore membrane, the activity of the HIV-1 protease was detected and quantified by monitoring the change in the ionic current passing through the nanopore, which is due to the cleavage of the peptide by the HIV-1 protease. The method was sensitive (detected HIV-1 protease at pico- to nanomolar levels) and selective (other proteases did not interfere with the detection).
A HIV-1 protease substrate peptide with a sequence of FFSQNYPIVQ (>98% pure) was purchased from Biomatik Corporation (Wilmington, Del.), while the HIV-1 protease was ordered from BioVendor Lab (Brno, Czech Republic). All the other chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.). The peptide, protease, and chemicals were dissolved in HPLC-grade water (ChromAR, Mallinckrodt Baker). The stock solutions of the peptide and the protease were prepared at 10 mM, and 300 μg/mL, respectively, and were kept at −80° C. before and after use. The electrolyte used contained 1.0 M NaCl buffered with 1 mM EDTA and 1 mM NaH2PO4, with the pH adjusted to 4.7 using H3PO4 solution. Lipid 1,2-diphytanoylphosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, Ala.). Teflon film (25 μm thick) was purchased from Goodfellow (Malvern, Pa.).
To prepare the protein nanopore, the mutant αHL M113F gene was constructed by site-directed mutagenesis (Mutagenex, Piscataway, N.J.) with a wild-type αHL gene in a T7 vector (pT7-αHL). The mutant αHL monomers were first synthesized by coupled in vitro transcription and translation (IVTT) using the E. Coli T7 S30 Extract System for Circular DNA from Promega (Madison, Wis.). Subsequently, they were assembled into homoheptamers by adding rabbit red cell membranes and incubating for 1-2 hours. The heptamers were then purified by SDS-polyacrylamide gel electrophoresis and stored in aliquots at −80° C.
A bilayer of 1,2-diphytanoylphosphatidylcholine was formed on an aperture (150 μm) in a Teflon septum that divided a planar bilayer chamber into cis and trans compartments, such as shown above
Data were analyzed with the following software: pClamp 10.3 (Molecular Devices), Origin 8.0 (Microcal, Northampton, Mass.), and SigmaPlot 12.0 (Systat Software Inc., San Jose, Calif.). Conductance values were obtained from the amplitude histograms after the peaks were fit to Gaussian functions. Values of τon and τoff for the peptide events were obtained from the open state (1) and close state (0) dwell time histograms, respectively by fitting the distributions to single exponential functions by the Levenberg-Marquardt procedure. The event frequency (f) was calculated by using the equation f=1/τon.
Initial experimentation was carried out at an applied potential bias of −40 mV in an electrolyte solution comprising 1 M NaCl, 1 mM EDTA and 1 mM NaH2PO4 (pH 4.7). This voltage has been demonstrated appropriate for nanopore peptide analysis, while pH 4.7 is the optimum solution pH for the detection of HIV-1 protease. As mentioned above, the nanopore sensing element used was a mutant α-hemolysin (αHL) protein, (M113F)7, while a peptide having a sequence of FFSQNYPIVQ was employed as the substrate. It has been shown that the αHL (M113F)7 protein could provide an enhanced resolution (e.g., prolonged residence time) for (bio-)molecule recognition compared with that observed with the wild-type αHL pore. Note that in the substrate design, two additional Phe amino acids were added to the sequence of a well-known HIV-1 protease substrate, SQNYPIVQ, for the purpose of creating two cleavage segments (i.e., FFSQNY and PIVQ) with different lengths. Unlike various conventional enzyme assays which detect the enzyme activity predominantly based on the signal decrease in the substrate, this nanopore sensor measured the HIV-1 protease activity based on the signal increase in the substrate degradation products. One significant advantage of such a sensor design strategy is that other interfering proteases (i.e., false positives) could be differentiated from the target HIV-1 protease if they cleave the peptide substrate at different positions, thus improving the sensor accuracy and selectivity. Since the molecular size (with dimensions of 45×23×25 Å) of the HIV-1 protease (a 99 amino acid aspartyl protease that functions as a homodimer with only one active site) is larger than that of the protein pore transmembrane domain (20 Å diameter), it could not enter the nanopore and hence could not produce current blockage events that might interfere with the identification of the target peptide(s) (See
Referring to
Upon addition of HIV-1 protease to the solution, two new types of blockage events having mean residual currents of −9.5 pA, and −18.0 pA were observed (labeled as “type 2” and “type 3” events in
It should be noted that since the smaller amplitude blockage events of the substrate degradation products partially overlapped with the background current spikes (˜one event per ten seconds) of the (M113F), pore, only the larger amplitude new type of (i.e., type 2) events were included in the data analysis of the event frequency. The detection limit (defined as the concentration corresponding to three times the standard deviation of a blank signal) for HIV-1 protease in a 1 hour enzymatic reaction was 0.47 ng/mL (equivalent to 47 pM).
To examine whether sensitive detection of HIV-1 protease could be achieved rapidly, the effect of the monitoring time (i.e., the reaction time) on the detection limit for protease detection was systematically studied. It should be noted that, in a proteolytic reaction, the instantaneous concentrations and hence the event frequencies of the substrate and the degradation products vary with the reaction time until all the substrate is degraded (
Hence, unlike the conventional nanopore sensing, where the event frequency is used as a parameter in the dose-response curve, the number of events (i.e., event counts) was used instead in this reaction time effect study. One advantage of replacing the event frequency with the number of events in the data analysis is that this approach can remedy the small event frequency issue, which is due to the low concentration of the degradation products at the early stage of an enzymatic reaction (especially for a reaction at a low concentration of protease), so that a long recording time is needed to collect enough events for the statistical analysis.
To demonstrate the application of the nanopore sensor in the analysis of samples resembling those relevant for clinical analysis, two samples were examined. One sample contained only HIV-1 protease, while the other contained a mixture of human serum album (HSA) and HIV-1 protease (note that HSA is a dominant protein in human blood). Similar to the HIV-1 protease, due to its larger molecular size (with approximate dimensions of 80×80×30 Å) than the trans opening of the αHL protein pore (˜20 Å), HSA cannot enter the nanopore and hence cannot produce current blockage events (
A new type of biosensor was developed to measure the activity of trypsin based on the enzymatic reactions between β-amyloid (10-20) and trypsin in an α-hemolysin nanopore. Given its advantages of real time, label-free and low-cost analysis, the developed nanopore sensor design strategy should find useful applications in the development of stochastic sensors for other proteases of medical, pharmaceutical, and biological importance.
Trypsin is a serine protease with 223 amino acid residues (M.W.=23.8 kDa), which is the most important digestive enzyme produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. Pancreatic trypsin production can be adversely affected by pathologies (e.g., pancreatitis), resulting in organ damage and release of enzyme into the blood. It is well known that the trypsin level is increased with some types of pancreatic diseases. Therefore, rapid and sensitive methods for trypsin detection, activity assay, and inhibitor screening are highly desired for the efficient diagnosis and treatment of pancreatic diseases.
The principle for nanopore detection of trypsin is shown in
β-amyloid (10-20) peptide, the trypsin substrate, with a sequence of YEVHHQKLVFF was obtained from American Peptide Company (Sunnyvale, Calif.), while peptides YEVHHQK and LVFF (i.e., two substrate degradation fragments) were obtained from Biomatik Corporation (Wilmington, Del.). All the other chemicals were bought from Sigma-Aldrich (St. Louis, Mo.). Trypsin, peptides, and all the other chemicals were dissolved in HPLC-grade water (ChromAR, Mallinchkrodt Baker). Stock solutions of the peptides were prepared at 0.5 mM each, while that of trypsin was 100 μg/ml. All these solutions were kept at −20° C. before and after use. The electrolyte used contained 1.0 M NaCl buffered with 1 mM Tris, with the pH adjusted to 7.5 using HCl. Lipid 1,2-diphytanoylphosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, Ala.). Teflon film (25 μm thick) was purchased from Goodfellow (Malvern, Pa.).
Engineered α-hemolysin (αHL) (M113F)7 protein nanopores were produced as mentioned above. Briefly, the mutant αHL M113F gene was constructed by site-directed mutagenesis (Mutagenex, Piscataway, N.J.) with a wild-type αHL gene in a T7 vector (pT7-αHL). The mutant αHL monomers were first synthesized by coupled in vitro transcription and translation using the E. coli T7 S30 Extract System for Circular DNA from Promega (Madison, Wis.). Subsequently, they were assembled into homoheptamers by adding rabbit red cell membranes and incubating for 1-2 hours. The heptamers were then purified by SDS-polyacrylamide gel electrophoresis and stored in aliquots at −80° C.
A bilayer of 1,2-diphytanoylphosphatidylcholine was formed on an aperture (150 μm) in a Teflon septum that divided a planar bilayer chamber into cis and trans compartments using the method of M. Montal and P. Mueller, Proc. Nat. Acad. Sci., 1972, 69, 3561-3566. Unless otherwise noted, all the experiments were performed under a series of symmetrical buffer conditions with a 1 M NaCl and 10 mM Tris (pH 7.5) at 24±1° C. The αHL protein was added to the cis compartment, which was connected to “ground”, while the peptide substrate and trypsin were added to the trans compartment. The final concentration of the αHL proteins used for the single channel insertion was 0.2-2.0 ng/ml. Currents were recorded with a patch clamp amplifier (Axopatch 200B, Molecular Devices; Sunnyvale, Calif., USA). Currents were low-pass filtered with a built-in four-pole Bessel filter at 5 KHz and sampled at 50 KHz by a computer equipped with a Digidata 1440A converter (Molecular Devices).
Data were analyzed with the following software: pClamp 10.3 (Molecular Devices), Origin 8.0 (Microcal, Northampton, Mass.), and SigmaPlot 12.0 (Systat Software Inc., San Jose, Calif.). Conductance values were obtained from the amplitude histograms after the peaks were fit to Gaussian functions. Values of inter-event interval (τon) and residence time (Toff) for the peptide events were obtained from the open state (1) and close state (0) dwell time histograms, respectively by fitting the distributions to single exponential functions by the Levenberg-Marquardt procedure. At least three separate experiments were carried out for each sample.
Initial experiments were carried out at an applied potential bias of −40 mV in 1 M NaCl solution buffered with 10 mM Tris (pH 7.5). The mutant αHL (M113F)7 protein nanopore was used as the stochastic sensing element, while β-amyloid (10-20) peptide (sequence: YEVHHQKLVFF, 5 μM) was employed as the substrate. It is well known that trypsin cuts peptide bonds after arginine or lysine amino acid residues, while the αHL (M113F)7 protein pore has been shown to offer an improved sensor resolution/sensitivity (e.g., prolonged event residence time for the analytes) over the wild-type αHL pore. Specifically, at this applied potential bias, all the three event parameters (i.e., residence time, frequency, and amplitude) for peptide translocation in the αHL nanopore had relatively large values; further, the open αHL channel was quiet without transient background current modulations. The experimental results are summarized in
The experiments were performed at −40 mV in 1 M NaCl solution buffered with 10 mM Tris (pH 7.5) using the mutant α-hemolysin protein (M113F)7 pore. The concentration of the substrate β-amyloid (10-20) was 5 μM. It is apparent that, with the β-amyloid (10-20) peptide alone in the solution, only one type of events was observed. These events had a mean residence time of ˜0.31 s and a mean residual current of approximately −2.25 pA. In contrast, after addition of trypsin to the substrate-containing solution, two types of new events with much smaller residence times and amplitudes appeared. These two types of new events had similar residence time (˜1.1 ms), but presented significantly different residual currents (˜5 pA vs.˜−12 pA), allowing them to be well separated and differentiated. Furthermore, the experiments showed that the frequencies of these two types of events increased with the recording time and with the increased concentration of added trypsin, a clear indication that they were attributed to the products formed by the trypsin's cleavage of the β-amyloid (10-20) peptide, i.e., YEVHHQK and LVFF, respectively. This result not only agreed very well with the fact that the spherical molecular diameter (38 Å) of trypsin is larger than that (20 Å) of the αHL transmembrane domain so that it cannot enter the pore, but also suggest that trypsin would not be denatured and pulled through the nanopore under the experimental condition, i.e., at a small applied voltage bias of −40 mV.
Under the same current experimental conditions (i.e., α-hemolysin (M113F)7 pore, pH 7.5, −40 mV applied voltage bias), a series of experiments was carried out to investigate the proteolytic cleavage of the substrate peptide β-amyloid (10-20) by trypsin, with the concentration of the substrate constant (5 μM), but varied the trypsin concentration (ranging from 5 to 50 ng/mL). Similar to the observation described above, in addition to the substrate events, two new types of current modulations were identified in all of the different trypsin concentration situations, providing further evidence that these events were attributed to the substrate degradation products. It should be noted that, in principle, the two substrate cleavage products should have identical concentrations. However, due to the length and structure difference in these two peptide fragments, the event frequencies for their translocation in the nanopore might vary greatly. Since the LVFF events were significantly more frequent than the YEVHHQK events, for convenience, only LVFF events were included in the data analysis. It should be noted that, one advantage of obtaining enzyme kinetic information based on the substrate degradation product signal instead of the substrate signal as used in various conventional enzyme assays is that this new strategy permits the target protease to be differentiated from other potential interfering proteases if they cleave the peptide substrate at different positions, and hence improved sensor selectivity and accuracy could be accomplished. Furthermore, in a proteolytic reaction, the instantaneous concentrations and hence the event frequencies of the degradation products vary with the reaction time until all the substrate is degraded. Therefore, unlike conventional nanopore sensing, where the event frequency is used as a parameter in the dose-response curve, the number of event occurrences (i.e., event counts) was used in this investigation instead. One advantage of utilizing event counts instead of the event frequency in the data analysis is that this approach can significantly shorten the experimental recording time. Due to the low concentration of the degradation products at the early stage of an enzymatic reaction (especially for a reaction with a low concentration of protease), a long recording time is required to collect enough events for statistical analysis of the event frequency.
The existence of Ca2+ ions in the solution can benefit the process of proteolytic reaction catalyzed by trypsin. Trypsin itself is a protein, and is capable of digesting itself. However, this autolysis process (more commonly known as self-digestion) can be prevented by Ca2+ ions (due to their binding to trypsin). In addition, Ca2+ ions can also promote the formation of active trypsin from trypsinogen, the protein produced in its inactive form within the pancrease of humans. To examine whether the existence of Ca2+ ions in the electrolyte solution could improve the nanopore sensor sensitivity for trypsin analysis, the effect of Ca2+ ions on the trypsin cleavage of the β-amyloid (10-20) peptide was further investigated. Experimental results, summarized in
Numerous methods have been utilized to study trypsin activity, including gel electrophoresis, gelatin-based film techniques, colorimetry, chemiluminescence, fluorescence, and electrochemical methods. Obviously, if more frequent events are observed for the substrate cleavage products, a stronger protease activity could be expected. In order to measure the activity of trypsin by nanopore analysis, a modified version of the universal protease activity assay was developed. Briefly, peptide YEVHHQK, one of the cleavage products of the substrate β-amyloid (10-20) peptide, was custom synthesized, and the dose-response curve (event counts vs. peptide concentration), shown in
To demonstrate the feasibility of utilizing the nanopore sensing platform of this invention as a screening tool for protease inhibitors, trypsin cleavage of the β-amyloid (10-20) peptide was further investigated in the presence of the trypsin inhibitor from bovine pancreas (TIBP), a small globular protein with 58 amino acid residues (M.W.=6.5 kDa). In principle, TIBP would bind to trypsin, thus inhibiting trypsin cleavage of β-amyloid (10-20) peptide. Therefore, compared with the situation without trypsin inhibitor, the presence of TIBP in the solution would lead to a decrease in the event frequency of the substrate cleavage products.
Thus the biosensor of this invention measured the activity of trypsin by monitoring the enzymatic reactions between the substrate and trypsin in an α-hemolysin nanopore. Given its advantages of real time, label-free and low-cost analysis, the nanopore sensing strategy should find useful applications in the development of stochastic sensors for other proteases of medical, pharmaceutical, and biological importance.
A label-free real-time nanopore sensing method and system for the detection of anthrax lethal factor, a component of the anthrax toxin, was prepared and tested by using a complementary single-stranded DNA as a molecular probe.
DNA samples of standard purification (desalting) were purchased from Intergrated DNA Technologies (Coralville, Iowa). All the other chemicals were ordered from Sigma-Aldrich (St. Louis, Mo.). All of the DNA samples and chemicals were dissolved in HPLC-grade water (ChromAR, Mallinckrodt Baker). All the stock solutions of DNA polymers were prepared at 5 mM each, and kept at −20° C. before and after use. Three electrolyte solutions were used in this work, which contained 0.15/1.0/3.0 M NaCl buffered with 10 mM Trizma base, with the pH adjusted to 7.5 using hydrochloride acid. Lipid 1,2-diphytanoylphosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, Ala.). Teflon film (25 um thick) was purchased from Goodfellow (Malvern, Pa.).
The mutant αHL M113F gene was constructed by site-directed mutagenesis (Mutagenex, Piscataway, N.J.) with a wild-type αHL gene in a T7 vector (pT7-αHL). The mutant αHL monomers were first synthesized by coupled in vitro transcription and translation (IVTT) using the E. Coli T7 S30 Extract System for Circular DNA from Promega (Madison, Wis.). Subsequently, they were assembled into homoheptamers by adding rabbit red cell membranes and incubating for 1-2 hours. The heptamers were then purified by SDS-polyacrylamide gel electrophoresis and stored in aliquots at −80° C.
A bilayer of 1,2-diphytanoylphosphatidylcholine was formed on an aperture (150 μm) in a Teflon septum that divided a planar bilayer chamber into cis and trans compartments. The formation of bilayer was achieved using the Montal et al. method discussed above. Unless otherwise noted, all the experiments were performed under symmetrical buffer conditions with a 2.0 mL solution comprising 1 M NaCl, and 10 mM Tris.HCl (pH 7.5) at 26±1° C. Both the αHL protein and the DNA polymers were added to the cis compartment, which was connected to “ground”. The final concentration of the αHL proteins used for the single channel insertion was 0.2-2.0 ng·mL−1. The transmembrane potential, which was applied with Ag/AgCl electrodes with 3% agarose bridges containing 3 M KCl, was +180 mV, unless otherwise noted. A positive potential indicates a higher potential in the trans chamber of the apparatus. Currents were recorded with a patch clamp amplifier (Axopatch 200B, Molecular Devices; Sunnyvale, Calif., USA). They were low-pass filtered with a built-in four-pole Bessel filter at 5 KHz and sampled at 50 KHz by a computer equipped with a Digidata 1322A/D converter (Molecular Devices).
Data were analyzed with the following software: pClamp 10.3 (Molecular Devices), Origin 8.0 (Microcal, Northampton, Mass.), and SigmaPlot 12.0 (Systat Software Inc., San Jose, Calif.). Conductance values were obtained from the amplitude histograms after the peaks were fit to Gaussian functions. The values of τon (the mean interevent interval) and τoff (the mean residence time) for DNA polymers were obtained from the dwell time histograms by fitting the distributions to single exponential functions by the Levenberg-Marquardt procedure. Thermodynamics of hairpin folding and DNA hybridization was obtained from the DINAMelt web server.
As shown in
To demonstrate this concept, a characteristic 20-base gene segment (SEQ ID NO:1: 5′-GGATTATTGTTAAATATTGA-3′) of anthrax lethal factor (aLF), a component of the anthrax toxin, was employed as the target pathogen molecule, while an engineered version of the αHL protein, (M113F)7, was used as the nanopore sensing element. The initial experiments were carried out at an applied potential bias of +120 mV and using a 20-base ssDNA (SEQ ID NO:2: 5′-TCAATATTTAACAATAATCC-3′) as the molecular probe, which can form blunt-ended double-stranded DNA with the target analyte. The experimental results, summarized in
As the aLF sample alone also only produced short-lived events with a mean residence time of 0.31±0.01 ms, the three-order increase in the event residence time suggests that the cDNA probe and the target aLF indeed formed DNA duplexes. According to the theoretical prediction (using the DINAMelt web server), both the probe and target DNA molecules could form thermodynamically stable hairpin loop structures (the Tm values were 30.9° C. and 34.9° C., respectively). The event residence times of these DNA hairpins were not significantly different from those of the well-studied ssDNA molecules with the same length; so the experimental results suggest that the closed states of these hairpin loop structures could be rapidly unfolded and driven through the nano-channel under our experimental conditions. As the target aLF DNA has a slightly larger folding free energy (ΔG=−0.72 kcal/mol vs. −0.42 kcal/mol), and hence more stable than the cDNA probe, it is not unreasonable that the aLF sample is more difficult to be unfolded, thus producing events with a longer mean residence time than the cDNA probe (0.31 ms vs. 0.20 ms). In contrast, when both the cDNA probe and the target aLF monomers are present in the solution, they would be able to form a very stable fully-matched DNA duplex with ΔG of −12.2 kcal/mol. Hence, significantly longer residence time events were observed.
These long-lived dsDNA events also exhibited sub-state current modulations, a clear indication that they might be attributed to the unzipping and translocation of the DNA duplex through the αHL channel. This interpretation was further supported by the voltage dependence study, where the mean residence time of the long-lived events decreased as the applied potential bias increased (
To study the aLF sensor selectivity, two other DNA samples, aLF-1 and aLF-2, were examined at +120 mV. These two samples had sequences of 5′-GGATTATTGTGAAATATTGA-3′ (SEQ ID NO:3), and 5′-GGATTATGGTGAAATATTGA-3′ (SEQ ID NO:4), respectively, which were different from the target analyte aLF (SEQ ID NO:4: 5′-GGATTATTGTTAAATATTGA-3′) only by a single base, and two bases (note that the mismatch portion are highlighted). The experimental results showed that the event mean residence times of the aLF-1 and aLF-2 samples were 342±35 ms and 57.4±2.5 ms, respectively. These values were about two-folds and ten-folds smaller than the residence time of the target aLF sample. The results are not unreasonable considering that the aLF-1/aLF-2 and the cDNA probe are able to form double-stranded DNA having single base-pair/two base-pair mismatches, which are less stable than the fully-matched aLF-cDNA duplexes, thus needing less time to be unzipped by the nanopore. This interpretation is supported by the predicted theoretical hybridization free energy between these DNA samples and the cDNA probe (using the DINAMelt web server), which were −12.2 kcal/mol, −10.9 kcal/mol, and −9.6 kcal/mol for aLF, aLF-1, and aLF-2, respectively. As added multiple-base mismatch controls, two additional DNA polymers A20 (SEQ ID NO:5: AAAAAAAAAAAAAAAAAAAA) and T20 (SEQ ID NO:6: TTTTTTTTTTTTTTTTTTTT) were also examined. Their event residence times were 11.7±0.2 and 0.12±0.01 ms, respectively, which was in agreement with our observation that with an increase in the number of the base-pair mismatches of the dsDNA sample, the DNA duplex became less stable, leading to a decrease in the event mean residence time (
To validate applicability of the nanopore sensor to samples resembling those relevant for clinical analysis, two samples were initially examined with the (M113F)7 protein pore in the presence of the cDNA probe and under the symmetric electrolyte condition. One sample contained human serum album (HSA), while the other contained a mixture of aLF and HSA (note that HSA is the dominant protein in human blood). As shown in
In summary, a rapid and sensitive nanopore sensing method for the label-free real-time detection of anthrax lethal factor was developed. By using an unmodified complementary single-stranded DNA as a molecular probe, and monitoring the hybridization interaction between a target single-stranded aLF gene segment and the cDNA probe, picomolar concentrations of aLF DNA could be detected in approximately 1 minute. Further, the method is selective, and other ssDNA molecules including those differing by only a single base will not interfere with the detection of the target aLF DNA. This sequence-specific detection approach should find useful application in the development of nanopore sensors for the detection of other pathogens.
The invention thus provides a real time, label-free and low-cost biomarker analysis nanopore sensor design useful in applications in the development of stochastic sensors for medical, pharmaceutical, and biological importance. Given the high sensitivity of the method and its potential to discriminate the target biomarker from false positives, the nanopore sensor design strategy can be used for stochastic sensors for various biomarker, especially those have potential clinical values for disease diagnosis or prognosis, and have become priority targets for new pharmaceuticals.
The invention illustratively disclosed herein suitably may be practiced in the absence of any element, part, step, component, or ingredient which is not specifically disclosed herein.
While in the foregoing detailed description this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.
This application claims the benefit of U.S. Provisional Patent Application, Ser. No. 61/952,554, filed on 13 Mar. 2014. The co-pending Provisional Patent Application is hereby incorporated by reference herein in its entirety and is made a part hereof, including but not limited to those portions which specifically appear hereinafter.
| Number | Date | Country | |
|---|---|---|---|
| 61952554 | Mar 2014 | US |
